Eukaryotic translation initiation factor 4GI (eIF4GI) is an essential protein that is the target for translational regulation in many cellular processes and viral systems. It has been shown to function in both cap-dependent and cap-independent translation initiation by recruiting the 40S ribosomal subunit to the mRNA cap structure or internal ribosome entry site (IRES) element, respectively. Interestingly eIF4GI mRNA itself has been reported to contain an IRES element in its 5 end that facilitates eIF4GI protein synthesis via a cap-independent mechanism. In HeLa cells, eIF4GI exists as several isoforms that differ in their migration in sodium dodecyl sulfate (SDS) gels; however, the nature of these isoforms was unclear. Here, we report a new cDNA clone for eIF4GI that extends the 5 sequence 340 nucleotides beyond the previously published sequence. The new extended sequence of eIF4GI is located on chromosome 3, within two additional exons immediately upstream of the previously published eIF4GI sequence. When mRNA transcribed from this cDNA clone was translated in vitro, five eIF4GI polypeptides were generated that comigrated in SDS-polyacrylamide gels with the five isoforms of native eIF4GI. Furthermore, translation of eIF4GI-enhanced green fluorescent protein fusion constructs in vitro or in vivo generated five isoforms of fusion polypeptides, suggesting that multiple isoforms of eIF4GI are generated by alternative translation initiation in vitro and in vivo. Mutation of two of the five in-frame AUG residues in the eIF4GI cDNA sequence resulted in loss of corresponding polypeptides after translation in vitro, confirming alternate use of AUGs as the source of the multiple polypeptides. The 5 untranslated region of eIF4GI mRNA also contains an out-of-frame open reading frame (ORF) that may down-regulate expression of eIF4GI. Further, data are presented to suggest that a proposed IRES embedded in the eIF4GI ORF is able to catalyze synthesis of multiple eIF4GI isoforms as well. Our data suggest that expression of the eIF4GI isoforms is partly controlled by a complex translation strategy involving both cap-dependent and cap-independent mechanisms.

A widespread common protein fold packages ssRNA in virus particles with diverse morphology and genomic structure.

No abstract

Eukaryotic translation initiation factor 4GI (eIF4GI)is an essential scaffolding protein required to recruit the 43 S complex to the 5-end of mRNA during translation initiation. We have previously demonstrated that eIF4GI protein expression is translationally regulated. This regulation is mediated by cis-acting RNA elements, including an upstream open reading frame and an IRES that directs synthesis of five eIF4GI protein isoforms via alternative AUG initiation codon selection. Here, we further characterize eIF4GI IRES function and show that eIF4GI is expressed from several distinct mRNAs that vary via alternate promoter use and alternate splicing. Several mRNA variants contain the IRES element. We found that IRES activity mapped to multiple regions within the eIF4GI RNA sequence, but not within the 5-UTR per se. However, the 5-UTR enhanced IRES activity in vivo and played a role in initiation codon selection. The eIF4GI IRES was active when transfected into cells in an RNA form, and thus, does not require nuclear processing events for its function. However, IRES activity was found to be dependent upon the presence, in cis, of a 5 m 7 guanosine-cap. Despite this requirement, the eIF4GI IRES was activated by 2A protease cleavage of eIF4GI, in vitro, and retained the ability to promote translation during poliovirus-mediated inhibition of capdependent translation. These data indicate that intact eIF4GI protein is not required for the de novo synthesis of eIF4GI, suggesting its expression can continue under stress or infection conditions where eIF4GI is cleaved.

Cryo–electron microscopy shows how the ribosome prepares for take-off to bypass a noncoding mRNA stretch.

Human enteroviruses and rhinoviruses rapidly and selectively abolish translation from cellular mRNA upon infection of susceptible cells. Expression of the poliovirus 2A protease (PV 2Apro) is sufficient to cause host translation shutoff through cleavage of elF4G (formerly p220, elF4 gamma) either directly or indirectly through activation of a cellular factor. Evidence exists for both direct and indirect cleavage mechanisms; however, factors presumed to participate in an indirect mechanism have not yet been purified or defined. Here we show that the dominant elF4G cleavage activity in lysates from infected HeLa cells was separable from PV 2Apro by size exclusion chromatography. 2Apro separated into two peak fractions which contained activity which cleaved a peptide substrate derived from the poliovirus polyprotein. These peak 2Apro fractions did not cleave elF4G or an elF4G-derived peptide, as expected, due to the poor efficiency of direct cleavage reactions. Conversely, fractions which contained peak elF4G cleavage activity and only trace amounts of 2Apro efficiently cleaved a peptide substrate derived from the previously mapped elF4G cleavage site and also cleaved a peptide derived from the poliovirus 1D2A region. The dominant elF4G cleavage activity was highly purified through four chromatography steps and found to be devoid of all traces of 2Apro or its precursors. Quantitation of 2Apro from lysates of infected cells showed that during infections in HeLa cells, 2Apro does not reach molar excess over elF4G, as previously shown to be required for direct elF4G cleavage in vitro. Further, infection of HeLa cells in the presence of 2 mM guanidine-HCl, a potent inhibitor of viral RNA replication, suppressed accumulation of 2Apro and its precursor 2ABC below detectable levels but was unable to delay the onset of elF4G proteolysis in vivo. The elF4G cleavage activity was still easily detectable in in vitro assays using fractions from guanidine-treated cells. Thus, the data suggest that poliovirus utilizes two catalytic activities to ensure rapid cleavage of elF4G in vivo. Although it was not directly measurable here, 2Apro likely does cleave a portion of elF4G in cells. However, the data suggest that a cellular factor which can be activated by small quantities of 2Apro constitutes the bulk of the elF4G-specific cleavage activity in infected cells and is responsible for the rapid and efficient elF4G cleavage activity observed in vivo.

Cleavage of eukaryotic translation initiation factor 4GI (eIF4GI) is required for shutoff of host cell translation during poliovirus (PV) infection ofpro , two distinct C-terminal cleavage fragments of eIF4GI were detected. These C-terminal cleavage fragments of eIF4GI were purified from infected cells, and a new eIF4GI cleavage site was mapped to a unique site 43 amino acids upstream of the known 2A pro cleavage site. Further, eIF4GI cleavage in vivo could be blocked by addition of zVAD to PV-guanidine infections. zVAD is a broad-spectrum caspase inhibitor which had no effect on 2A pro cleavage activity or PV polyprotein processing. Lastly, similar types of eIF4Gase cleavage activities were also detected in uninfected cells under various conditions, including early apoptosis or during cell cycle transit. The data suggest that the same types of eIF4GI cleavage activities which are generated in PV-infected cells can also be generated in the absence of virus. Taken together, the data support a model in which multiple cellular activities process eIF4GI in PV-infected cells, in addition to 2A pro .

The nucleotide sequences of the F and M2 mRNAs of strain A51908 of bovine respiratory syncytial virus (BRSV) were determined by sequencing cDNA of an intracellular dicistronic mRNA. Comparison of the F mRNA sequence with those of other BRSV strains showed that there was extensive sequence identity at both the nucleotide (95% identity) and amino acid (94 % identity) levels. Alignment of the nucleotide and encoded amino acid sequences of M2 mRNA of BRSV with those of human respiratory syncytial virus (HRSV) M2 mRNA showed 69% identity at the nucleotide level and 80% identity at the amino acid level. The general features ofBRSV F and M2 proteins are similar to those described previously for the HRSV proteins. The M2 mRNA of BRSV also contained a second internal, overlapping open reading frame (ORF) similar to one reported for HRSV. The predicted products of the second ORFs of BRSV and HRSV shared 43 % amino acid identity. As described for HRSV, the T-terminal end of the M2 mRNA overlaps with the 5' end of the L gene by 68 nucleotides. The identity between the N-terminal regions of the L proteins of BRSV and HRSV is 75 %. In addition, the intergenic sequence of the F-M2 gene junction of BRSV was determined.